Sensitive and Quantitative MS-bases Glycomic Mapping Platform

基于 MS 的灵敏定量糖组图谱平台

基本信息

  • 批准号:
    10019565
  • 负责人:
  • 金额:
    $ 28.4万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2014
  • 资助国家:
    美国
  • 起止时间:
    2014-09-15 至 2021-08-31
  • 项目状态:
    已结题

项目摘要

Glycomics has emerged as an interesting yet challenging area of research in biology. Glycans function in numerous important biological areas such as, but not limited to: the immune system, cell development, cell differentiation/adhesion, host-pathogen interactions, protein signaling, and protein stabilization. Abnormal glycosylation has been associated with several diseases including cancer, cystic fibrosis, and osteoarthritis. Glycomics/glycoproteomics studies aim to quantify and characterize glycan structures (including linkage and positional isomers), protein attachment sites, and the protein’s identity. Approximately 50% of mammalian proteins are glycosylated but their abundance is rather low compared to non-glycosylated proteins. Furthermore, numerous glycans can occupy the same glycan attachment site on a protein; that is the same protein pool can have several different types of glycans attached to the same site, each with a potentially different function or a particular activity. Protein glycans are divided into two classes based on their amino acid attachment sites: asparagine for N-glycans and threonine, serine, and tyrosine for O-glycans. A strategy that has been successfully employed to investigate N-glycans in cells is to release or separate the glycans from proteins with the enzyme PNGase F, and study the global glycan composition of a sample. A drawback to this approach is that, so called, native glycans possess low ionization efficiencies which make their analysis by mass spectrometry quite difficult; however, this sensitivity issue can be overcome by permethylating glycans. Glycans have many isomers which can make their accurate analysis by LC-MS/MS difficult if the isomers cannot be resolved. This proposal demonstrates that we are able to separate permethylated glycan isomerss with a heated PGC column before mass spectrometry analysis (Aim 1), resulting in an extremely sensitive assay to accurately characterize and quantitate glycan isomers in biological samples. Although the separation of isomeric glycans has been previously reported, prior studies only resolved native and reducing end labeled glycan structures. Owing to the fact that permethylated glycans exhibit ionization efficiencies at least two orders of magnitude higher than the aforementioned structures, the importance of the increase in sensitivity, for the detection of structures at physiological concentrations, that accompanies isomeric separation of permethylated glycans (Aim 1) cannot be overstated. To overcome the variation in ionization efficiency between LC-MS samples, we have successfully permethylated glycans with various stable isotope combinations to achieve unprecedented quantitative glycan comparisons across samples derived from cell culture experiments, biological fluids, and biological tissues. Through the implementation of our multi-level isotopic labeling strategies (metabolic 15N labeling, 18O reducing end labeling and multiplex permethylation), the number of potential multiplexed samples can be increased from eight, the previous maximum, to 16 and 32 for biological fluid and tissue samples and cell culture samples, respectively (Aim 2). High throughput isomeric characterization glycans derived from biological samples can be attained by combining the methods described in Aims 1 and 2. While PNGase F is used extensively to release N-glycans from proteins, no such enzyme exists or has been discovered for O-glycans. We have developed a rapid method, RAIDR (Rapid Ammonium hydroxide Isobutyric acid O-glycan Deglycosylation Reaction), for selectively releasing O-glycans; RAIDR leaves the protein and N-glycans unscathed which allows for compatible downstream analyses (Aim 3). In addition to improving LC-MS analytical methods, we are also proposing the addition of graphene nanosheets and carbon nanoparticles to MALDI matrices for enhanced sample preparation, cleanup, and an increase in the ionization efficiencies of both native and permethylated glycans (Aim 4). Mass spectrometry based experiments can generate a tremendous amount of data that is cumbersome to analyze manually. There are numerous well-known proteomic software packages available but few that can comprehensively analyze glycomic datasets. We have developed MultiGlycan to analyze glycomic datasets and intend to expand its functionalities (Aim 5) to handle glycan isomers (Aim 1), multiplexed permethylated glycans (Aim 2), O-glycans (Aim 3), and glycans analyzed with MALDI-MS (Aim 4). The development of the proposed methods and algorithms will help us and collaborators to better understand the attributes and biomedical significance of glycan isomers in the development and progression of esophagus, breast, and liver cancer. We are also expecting the analytical tools and algorithms proposed here to be beneficial to other scientists who are interested in understanding the biological attributes of glycan isomers in other systems to benefit from.
糖组学已经成为生物学中一个有趣但具有挑战性的研究领域。聚糖在

项目成果

期刊论文数量(0)
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Yehia Mechref其他文献

Yehia Mechref的其他文献

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{{ truncateString('Yehia Mechref', 18)}}的其他基金

Quantitative Characterization of Glycopeptide Isomers
糖肽异构体的定量表征
  • 批准号:
    10331873
  • 财政年份:
    2019
  • 资助金额:
    $ 28.4万
  • 项目类别:
Quantitative Characterization of Glycopeptide Isomers
糖肽异构体的定量表征
  • 批准号:
    10540152
  • 财政年份:
    2019
  • 资助金额:
    $ 28.4万
  • 项目类别:
Sensitive and Quantitative MS-bases Glycomic Mapping Platform
基于 MS 的灵敏定量糖组图谱平台
  • 批准号:
    8787914
  • 财政年份:
    2014
  • 资助金额:
    $ 28.4万
  • 项目类别:
Sensitive and Quantitative MS-bases Glycomic Mapping Platform
基于 MS 的灵敏定量糖组图谱平台
  • 批准号:
    8927045
  • 财政年份:
    2014
  • 资助金额:
    $ 28.4万
  • 项目类别:
Sensitive and Quantitative MS-bases Glycomic Mapping Platform
基于 MS 的灵敏定量糖组图谱平台
  • 批准号:
    10318016
  • 财政年份:
    2014
  • 资助金额:
    $ 28.4万
  • 项目类别:
Sensitive and Quantitative MS-bases Glycomic Mapping Platform
基于 MS 的灵敏定量糖组图谱平台
  • 批准号:
    10697345
  • 财政年份:
    2014
  • 资助金额:
    $ 28.4万
  • 项目类别:
Sensitive and Quantitative MS-bases Glycomic Mapping Platform
基于 MS 的灵敏定量糖组图谱平台
  • 批准号:
    9120382
  • 财政年份:
    2014
  • 资助金额:
    $ 28.4万
  • 项目类别:

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